Calpain pp 195-206 | Cite as

Immunoblotting for Calpastatin Expression

  • Monica AvernaEmail author
  • Roberta De Tullio
Part of the Methods in Molecular Biology book series (MIMB, volume 1915)


Immunoblotting is a procedure routinely used to analyze calpastatin expression. However, immunoblotting alone may not be adequate for this task, since calpastatin isoforms can vary by tissue, can be modified by partial digestion, and can undergo posttranslational modifications. Here we describe a method for more precise evaluation of calpastatin expression by combining immunoblot analysis with an assay for the inhibitory activity of a single calpastatin species isolated by SDS-PAGE and protein elution from the gel.

Key words

Immunoblot Calpastatin Calpain Protein elution Ca2+-dependent proteolysis Calpastatin phosphorylation 



This work was supported by grants FRA2015 and FRA2016 from the University of Genoa to MA and RDT.


  1. 1.
    Takano J, Watanabe M, Hitomi K, Maki M (2000) Four types of calpastatin isoforms with distinct amino-terminal sequences are specified by alternative first exons and differentially expressed in mouse tissues. J Biochem 128:83–92CrossRefGoogle Scholar
  2. 2.
    Parr T, Jewell KK, Sensky PL, Brameld JM, Bardsley RG, Buttery PJ (2004) Expression of calpastatin isoforms in muscle and functionality of multiple calpastatin promoters. Arch Biochem Biophys 427:8–15CrossRefGoogle Scholar
  3. 3.
    Li S, Liang ZG, Wang GY, Yavetz B, Kim ED, Goldberg E (2000) Molecular cloning and characterization of functional domains of a human testis-specific isoform of calpastatin. Biol Reprod 63:172–178CrossRefGoogle Scholar
  4. 4.
    Parr T, Sensky PL, Bardsley RG, Buttery PJ (2001) Calpastatin expression in porcine cardiac and skeletal muscle and partial gene structure. Arch Biochem Biophys 395:1–13CrossRefGoogle Scholar
  5. 5.
    Raynaud P, Vignoles-Jayat C, Laforêt MP, Leveziel H, Amarger V (2005) Four promoters direct expression of the calpastatin gene. Arch Biochem Biophys 437:69–77CrossRefGoogle Scholar
  6. 6.
    Lee WJ, Ma H, Takano E, Yang HQ, Hatanaka M, Maki M (1992) Molecular diversity in amino-terminal domains of human calpastatin by exon skipping. J Biol Chem 267:8437–8442PubMedGoogle Scholar
  7. 7.
    De Tullio R, Sparatore B, Salamino F, Melloni E, Pontremoli S (1998) Rat brain contains multiple mRNAs for calpastatin. FEBS Lett 422:113–117CrossRefGoogle Scholar
  8. 8.
    Takano J, Kawamura T, Murase M, Hitomi K, Maki M (1999) Structure of mouse calpastatin isoforms: implications of species-common and species-specific alternative splicing. Biochem Biophys Res Commun 260:339–345CrossRefGoogle Scholar
  9. 9.
    Geesink GH, Nonneman D, Koohmaraie M (1998) An improved purification protocol for heart and skeletal muscle Calpastatin reveals two isoforms resulting from alternative splicing. Arch Biochem Biophys 356:19–24CrossRefGoogle Scholar
  10. 10.
    De Tullio R, Averna M, Stifanese R, Parr T, Bardsley RG, Pontremoli S, Melloni E (2007) Multiple rat brain calpastatin forms are produced by distinct starting points and alternative splicing of the N-terminal exons. Arch Biochem Biophys 465:148–156CrossRefGoogle Scholar
  11. 11.
    Averna M, Stifanese R, De Tullio R, Passalacqua M, Defranchi E, Salamino F, Melloni E, Pontremoli S (2007) Regulation of calpain activity in rat brain with altered Ca2+ homeostasis. J Biol Chem 282:2656–2665CrossRefGoogle Scholar
  12. 12.
    Averna M, De Tullio R, Salamino F, Minafra R, Pontremoli S, Melloni E (2001) Age-dependent degradation of calpastatin in kidney of hypertensive rats. J Biol Chem 276:38426–38432CrossRefGoogle Scholar
  13. 13.
    Takano E, Maki M, Mori H, Hatanaka M, Marti T, Titani K, Kannagi R, Ooi T, Murachi T (1988) Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis. Biochemistry 27:1964–1972CrossRefGoogle Scholar
  14. 14.
    Pontremoli S, Viotti PL, Michetti M, Salamino F, Sparatore B, Melloni E (1992) Modulation of inhibitory efficiency of rat skeletal muscle calpastatin by phosphorylation. Biochem Biophys Res Commun 187:751–759CrossRefGoogle Scholar
  15. 15.
    De Tullio R, Averna M, Salamino F, Pontremoli S, Melloni E (2000) Differential degradation of calpastatin by mu- and m-calpain in Ca2+-enriched human neuroblastoma LAN-5 cells. FEBS Lett 475:17–21CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Experimental Medicine (DIMES)—Biochemistry SectionUniversity of GenovaGenovaItaly
  2. 2.Centre of Excellence for Biomedical Research (CEBR)University of GenovaGenovaItaly

Personalised recommendations